Effects of single-base substitutions within the acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I
Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decrease specific RNA synthesis in vitro. These were within the region which is protected from DNAse I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.
- Research Organization:
- Dept. of Pathology, Univ. of Massachusetts Medical School, Worcester, MA 01605 (US)
- OSTI ID:
- 5115284
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 8:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
AMOEBA
GENOME MUTATIONS
RIBOSOMAL RNA
GENES
RNA POLYMERASES
ENZYME ACTIVITY
CLONE CELLS
DNA SEQUENCING
DNA-ASE
IN VITRO
MUTAGENS
TRANSCRIPTION
ANIMALS
CELL CULTURES
ENZYMES
ESTERASES
HYDROLASES
INVERTEBRATES
MICROORGANISMS
MUTATIONS
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTOZOA
RNA
SARCODINA
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550200* - Biochemistry